Our long-term goal of this R37 is to understand human B cell tolerance and its breakdown in autoimmunity. Our original aims sought to elucidate the role of receptor editing and antigen selection in human B cell tolerance (Aim 1); and to understand antigen-driven selection of SLE B cells with emphasis on the role of apoptotic cell antigens (Aim 2). To do so, we established high-dimension flow cytometry to precisely classify and purify human B cell subsets and identified new B cell and plasma cell subsets that provide the required cellular knowledge to address our questions. We also incorporated single cell generation of monoclonal antibodies and Next Generation Sequencing (NGS). We generated in excess of 20,000 heavy/light chain pairs from autoreactive B cells in SLE and HIV and more than 500 monoclonal antibodies; 3) performed NGS of 52 B cell samples purified from SLE, other autoimmune diseases, HIV and vaccinated healthy controls. These studies have largely answered the questions posed in our original aims as we elucidated the structural basis of different types of lupus autoantibodies, including anti-apoptotic cell antibodies, and demonstrated deficient receptor editing in autoimmune lupus B cells (Aim 1). The combination of single cell mAb analysis and NGS allowed us to demonstrate that while apoptotic cell antigens play an important role in the selection of autoimmune memory cells in SLE, other self-antigens must be dominant in shaping this compartment and regulating its contribution to the antibody secreting cell compartment in active disease (Aim 2). To further achieve our aims and better understand the antigenic, genetic and molecular mechanisms that regulate tolerance and license autoimmune B cells we are also pursuing the analysis of the human serum autoantibody proteome and have initiated integrated studies of the transcriptome and epigenome of autoimmune B cells. Combined these powerful tools enable comprehensive phenotypic, genetic and molecular analysis of human B cells, precise purification of carefully defined developmental subsets and high-throughput analysis of the cellular and secreted antibody repertoires. RELEVANCE (See instructions): Understanding B cell tolerance is essential to prevent and treat autoimmunity, allergy and transplant rejection. Our studies will continue to unravel the nature of the antigens that trigger and amplify autoimmunity and will also elucidate the genetic and molecular roadmaps that underlie B cell malfunction. The expected results should greatly enhance our ability to identify early disease and its triggers; identify biomarkers of autoimmune risk and disease progression: and generate new therapeutic targets.